Parallel RNA and DNA analysis after Deep-sequencing (PRDD-seq) reveals cell type specific lineage patterns in human brain

Abstract

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed Parallel RNA and DNA analysis after Deep-sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables the first-ever simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation (“birthdate”) in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an “inside-out” layer formation of excitatory neurons as seen in other species. In addition our analysis allows the first estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.Significance StatementStem cells and progenitors undergo a series of cell divisions to generate the neurons of the brain, and understanding this sequence is critical to studying the mechanisms that control cell division and migration in developing brain. Mutations that occur as cells divide are known as the basis of cancer, but have more recently been shown to occur with normal cell divisions, creating a permanent, forensic map of the clonal patterns that define the brain. Here we develop new technology to analyze both DNA mutations and RNA gene expression patterns in single cells from human postmortem brain, allowing us to define clonal patterns among different types of human brain neurons, gaining the first direct insight into how they form.

Application of single cell genomics to focal epilepsies: A call to action

“…For example, when a neuroglial progenitor has spontaneous DNA damage that escapes the DNA repair machinery, it accumulates variants that are unique to that cell and are passed down to its progeny as a lineage barcode (Figure 2). If the lineage‐defining clonal variants in a given tissue are known—typically through bulk or synthetic bulk WGS (143)—it is possible to trace back the developmental origin of cortical neurons or glia by identifying the variants that they share. The cell types and the fraction of cells carrying a specific somatic variant could serve as surrogate markers to infer the developmental timing of when a mutation occurred (11, 144).…”

“…The cell types and the fraction of cells carrying a specific somatic variant could serve as surrogate markers to infer the developmental timing of when a mutation occurred (11, 144). This technique is particularly powerful when combined with RNA sequencing, as it can draw a connection between specific cell types, their developmental origins, and their timeline of differentiation (143). Lineage tracing in the human brain has so far been mostly limited to the study of normal tissue (11, 143, 144), but it is easy to imagine how this transformative technology could elucidate the mechanisms of genetic focal epilepsies.…”

“…This technique is particularly powerful when combined with RNA sequencing, as it can draw a connection between specific cell types, their developmental origins, and their timeline of differentiation (143). Lineage tracing in the human brain has so far been mostly limited to the study of normal tissue (11, 143, 144), but it is easy to imagine how this transformative technology could elucidate the mechanisms of genetic focal epilepsies. Lineage tracing could shed light on the timing of when pathogenic somatic variants arise and the conditions under which they cause disease.…”

Profile of Christopher A. Walsh

“…More recently, Walsh has become increasingly interested in basic biology, resulting in work that addresses questions raised early in his career (1). "My Inaugural Article combines my interests in human genetic mutations that confer risk and in understanding the basic biology of the brain," he says.…”

“…Walsh, who holds appointments at Boston Children's Hospital and Harvard Medical School, is a Howard Hughes Medical Institute Investigator and was elected to the National Academy of Sciences in 2018. His Inaugural Article uses cutting-edge technology to address questions about cerebral cortex development (1). "It's about the sequence of formation of the cells that form our organ of consciousness," says Walsh.…”